Ethanol production from starch by a coimmobilized mixed culture system of Aspergillus awamori and Zymomonas mobilis

The production of ethanol from starch by a coimmobilized mixed culture system of aerobic and anaerobic microorganisms in Ca-alginate gel beads was investigated. The mold Aspergillus awamori was used as an aerobic amylolytic microorganism and an anaerobic bacterium, Zymomonas mobilis, as an ethanol producer. By controlling the mixing ratio of the microorganisms in the inoculum size, a desirable coimmobilized mixed culture system, in which the aerobic mycelia grew on and near the oxygen-rich surface of the gel beads while the anaerobic bacterial cells mainly grew in the oxygen-deficient central part of the gel beads, was naturally established under the aerobic culture conditions, and ethanol could be directly produced from starch by the system. The ethanol productivity by the system in flask culture was particularly affected by the shear stress (dependent on the shaking speed) which controlled the mycelial growth on the surface of the gel beads. Under optimum culture conditions in the flask culture, the glucose produced was instantly consumed, and was not observed in the culture broth; the final concentration of ethanol produced from 100 g/L starch was 25 g/L and the yield coefficient for ethanol, Y(pls), was 0.38. The ethanol productivity by the coimmobilized mixed culture system was compared with those by other various culture systems and the advantages of the system were clarified.     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001     

Production of ethanol directly from potato starch by mixed culture of Saccharomyces cerevisiae and Aspergillus niger using electrochemical bioreactor

When cultivated aerobically, Aspergillus niger hyphae produced extracellular glucoamylase, which catalyzes the saccharification of unliquified potato starch into glucose, but not when grown under anaerobic conditions. The Km and Vmax of the extracellular glucoamylase were 652.3 mg starch l-1 and 253.3 mg glucose l-1 min-1, respectively. In mixed culture of A. niger and Saccharomyces cerevisiae, oxygen had a negative influence on the alcohol fermentation of yeast, but activated fungal growth. Therefore, oxygen is a critical factor for ethanol production in the mixed culture, and its generation through electrolysis of water in an electrochemical bioreactor needs to be optimized for ethanol production from starch by coculture of fungal hyphae and yeast cells. By applying pulsed electric fields (PEF) into the electrochemical bioreactor, ethanol production from starch improved significantly: Ethanol produced from 50 g potato starch l-1 by a mixed culture of A. niger and S. cerevisiae was about 5 g l-1 in a conventional bioreactor, but was 9 g l-1 in 5 volts of PEF and about 19 g l-1 in 4 volts of PEF for 5 days.     

Conversion of dextrinized cassava starch into ethanol using cultures of Aspergillus awamori and Saccharomyces cerevisiae

Aspergillus awamori and Saccharomyces cerevisiae have been used to convert dextrinized cassava root flour into ethanol. A batch culture of the combined microorganisms produced 4.3% alcohol by weight from 15% cassava flour slurry in 39 h. Two-stage continuous fermentation was done using A. awamori in an airlift fermenter and yeast in a tower fermenter. A residence time of 12.5 h for the first stage resulted in 12.5% sugar concentration and a saccharification efficiency of 88%. A residence time of 5.6 h for the second stage gave an alcohol concentration of 5.3% alcohol and a starch-into-ethanol conversion efficiency of 72.5%.     

Improvement of polygalacturonase production at high temperature by mixed culture of Aspergillus niger and Saccharomyces cerevisiae

Catabolic repression in the synthesis of inducible enzymes by glucose, fructose, and intermediates of the glycolytic cycle has been observed in many microorganisms. In order to enhance the polygalacturonase (PG) production of Aspergillus niger GJ-2, Saccharomyces cerevisiae J-1 was inoculated to the medium at 12 h of culture, which resulted in a significant improvement of PG production. It was also found that maximum PG activity of 512.7 U/ml was obtained at 37 °C in the mixed culture, which was nearly twofold higher than that of the culture without the inoculation of S. cerevisiae J-1.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.     

Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

In a batch mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae, which could assimilate lactic acid, cell growth and kefiran production rates of L. kefiranofaciens significantly increased, compared with those in pure cultures. The kefiran production rate was 36 mg l(-1) h(-1) in the mixed culture under the anaerobic condition, which was greater than that in the pure culture (24 mg l(-1) h(-1)). Under the aerobic condition, a more intensive interaction between these two strains was observed and higher kefiran production rate (44 mg l(-1) h(-1)) was obtained compared with that under the anaerobic condition. Kefiran production was further enhanced by an addition of fresh medium in the fed-batch mixed culture. In the fed-batch mixed culture, a final kefiran concentration of 5.41 g l(-1) was achieved at 87 h, thereby attaining the highest productivity at 62 mg l(-1) h(-1). Simulation study considered the reduction of lactic acid in pure culture was performed to estimate the additional effect of coculture with S. cerevisiae. Slightly higher cell growth and kefiran production rates in the mixed culture than those expected from pure culture by simulation were observed. These results suggest that coculture of L. kefiranofaciens and S. cerevisiae not only reduces the lactic acid concentration by consumption but also stimulates cell growth and kefiran production of L. kefiranofaciens.     

Degradation of raw starch granules by alpha-amylase purified from culture of Aspergillus awamori KT-11

Raw-starch-digesting alpha-amylase (Amyl III) was purified to an electrophoretically pure state from the extract of a koji culture of Aspergillus awamori KT-11 using wheat bran in the medium. The purified Amyl III digested not only soluble starch but also raw corn starch. The major products from the raw starch using Amyl III were maltotriose and maltose, although a small amount of glucose was produced. Amyl III acted on all raw starch granules that it has been tested on. However, it was considered that the action mode of the Amyl III on starch granules was different from that of glucoamylase judging from the observation of granules under a scanning electron microscope before and after enzyme reaction, and also from the reaction products. Glucoamylase (GA I) was also isolated and it was purified to an electrophoretically pure state from the extract. It was found that the electron micrographic features of the granules after treatment with the enzymes were quite different. A synergistic effect of Amyl III and GA I was observed for the digestion of raw starch granules.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997     

Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

 Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l^-1 h^-1. No levan was produced even if a sucrose-based medium was used. Received: 20 April 1995/Received revision: 26 July 1995/Accepted: 13 September 1995     

Ethanol production from nonsterilized carob pod extract by free and immobilized Saccharomyces cerevisiae cells using fed-batch culture

The production of ethanol from carob pod extract by free and immobilized Saccharomyces cerevisiae cells in batch and fed-batch culture was investigated. Fed-batch culture proved to be a better fermentation system for the production of ethanol than batch culture. In fed-batch culture, both free and immobilized S. cerevisiae cells gave the same maximum concentration (62 g/L) of final ethanol at an initial sugar concentration of 300 g/L and F = 167 mL/h. The maximum ethanol productivity (4.4 g/L h) was obtained with both free and immobilized cells at a substrate concentration of 300 g/L and F = 334 mL/h. In repeated fed-batch culture, immobilized S. cerevisiae cells gave a higher overall ethanol concentration compared with the free cells. The immobilized S. cerevisiae cells in Ca-alginate beads retained their ability to produce ethanol for 10 days. (c) 1994 John Wiley & Sons, Inc.     

Degradation of phenol by a coimmobilized entrapped mixed culture

Summary The degradation of phenol by a defined mixed culture, consisting of Pseudomonas putida P8 and Cryptococcus elinovii H1, was studied. The microorganisms were entrapped either in 30 g·l^-1 calcium-alginate or in chitosan-alginate. Chitosan-alginate entrapment was suitable for a continuous culture. The coimmobilized mixed culture of Cryptococcus elinovii H1 which degrades phenol via an ortho pathway and of Pseudomonas putida P8 which uses the meta cleavage pathway was able to degrade high phenol concentrations up to 3.2 g·l^-1 in semicontinuous cultures. The degradation performance in continuous cultures could reach a maximum of 0.41 g·l^-1·h^-1 phenol. The mixed culture could be stored for up to six months without loss of phenol degradation capacity.Dedicated to Professor Dr. Dr. h. c. K. Esser on the occasion of his 65th birthday     

Ethanol production by Saccharomyces cerevisiae using lignocellulosic hydrolysate from Chrysanthemum waste degradation

Ethanol production derived from Saccharomyces cerevisiae fermentation of a hydrolysate from floriculture waste degradation was studied. The hydrolysate was produced from Chrysanthemum (Dendranthema grandiflora) waste degradation by Pleurotus ostreatus and characterized to determine the presence of compounds that may inhibit fermentation. The products of hydrolysis confirmed by HPLC were cellobiose, glucose, xylose and mannose. The hydrolysate was fermented by S. cerevisiae, and concentrations of biomass, ethanol, and glucose were determined as a function of time. Results were compared to YGC modified medium (yeast extract, glucose and chloramphenicol) fermentation. Ethanol yield was 0.45 g g^?1, 88 % of the maximal theoretical value. Crysanthemum waste hydrolysate was suitable for ethanol production, containing glucose and mannose with adequate nutrients for S. cerevisiae fermentation and low fermentation inhibitor levels.     

Ethanol production by immobilized Saccharomyces cerevisiae, Saccharomyces uvarum, and Zymomonas mobilis

Saccharomyces cerevisiae NRRL Y-2034, S, uvarum NRRL Y-1347, and Zymomonas mobilis NRRL B-806 each were separately immobilized in a Ca-alginate matrix and incubated in the presence of a free-flowing and continuous 1, 3, 5, 10, or 20% (w/w) glucose solution. In general, the yeast cells, converted 100percnt; of the 1, 3, and 5% glucose to alcohol within 48 h and maintained such a conversion rate for at least two weeks. The bacterium converted ca. 90% (w/w) of the 1, 3, and 5% glucose to alcohol continuously for one week. However, both the yeast and bacterium were inhibited in the highest glucose (20% w/w) solution. All of the immobilized cultures produced some alcohol for at least 14 days. Immobilized S. cerevisiae was the best alcohol producer of all of the glucose concentrations; the yeast yielded 4.7 g ethanol/100 g solution within 72 h in the 10% glucose solution. After 7-8 days in the 10% solution, S. cerevisiae produced ethanol at 100% of theoretical yield (5.0 g ethanol/100 g solution), with a gradual decrease in alcohol production by 14 days. Immobillized S. uvarum produced a maximum of 4.0 g ethanol/100 g solution within 2 days and then declined to ca. 1.0 g ethanol/100 g solution after 7 days continuous fermentation in the 10% glucose solution. Zymomonas mobilis reached its maximum ethanol production at 4 days (4.7 g/100 g solution), and then diminished similarly to S. uvarum. The development of a multiple disk shaft eliminated the problem both of uneven distribution of alginate-encapsulated cells and of glucose channeling within the continuous-flow fermentor column. This invention improved alcohol production about threefold for the yeast cells.     

Ethanol production in a hollow fiber bioreactor using Saccharomyces cerevisiae

Summary A new approach for continuous production of ethanol was developed using a Hollow fiber fermentor (HFF). Saccharomyces cerevisiae cells were packed into the shell-side of a hollow fiber module. Using 100 g/l glucose in the feed gave an optimum ethanol productivity, based on total HFF volume, of 40 g ethanol/l/h at a dilution rate of 3.0 h^-1. Under these conditions, glucose utilization was 30%. However, at 85% glucose utilization the productivity was 10 g ethanol/l/h. This compares to batch fermentor productivity of 2.1 g ethanol/l/h at 100% glucose utilization.